Inhibition of matrix metalloproteases by substituted biaryl oxobutyric acids

ABSTRACT

Matrix metalloprotease inhibiting compounds, pharmaceutical compositions thereof and a method of disease treatment using such compounds are presented. The compounds of the invention have the generalized formulas:                    
     wherein r is 0-2, T is selected from                    
     and R 40  is a mono- or bi-heterocyclic structure. 
     These compounds are useful for inhibiting matrix metalloproteases and, therefore, combating conditions to which MMP&#39;s contribute, such as osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulceration, proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa, conditions leading to inflammatory responses, osteopenias mediated by MMP activity, tempero mandibular joint disease, demyelating diseases of the nervous system, tumor metastasis or degenerative cartilage loss following traumatic joint injury, and coronary thrombosis from arteriosclerotic plaque rupture. The present invention also provides pharmaceutical compositions and methods for treating such conditions.

This application is a division of Ser. No. 08/856,693 filed May 15, 1997now U.S. Pat. No. 5,925,637.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to enzyme inhibitors, and more particularly, tonovel substituted biaryl oxobutyric acid compounds or derivativesthereof useful for inhibiting matrix metalloproteases.

2. Description of the Related Art

The matrix metalloproteases (a.k.a. matrix metalloendo-proteinases orMMPs) are a family of zinc endoproteinases which include, but are notlimited to, interstitial collagenase (a.k.a. MMP-1), stromelysin (a.k.a.proteoglycanase, transin, or MMP-3), gelatinase A (a.k.a. 72kDa-gelatinase or MMP-2) and gelatinase B (a.k.a. 95 kDa-gelatinase orMMP-9). These MMPs are secreted by a variety of cells includingfibroblasts and chondrocytes, along with natural proteinaceousinhibitors known as TRMPs tissue Inhibitor of MetalloProteinase).

All of these MMPs are capable of destroying a variety of connectivetissue components of articular cartilage or basement membranes. Each MMPis secreted as an inactive proenzyme which must be cleaved in asubsequent step before it is able to exert its own proteolytic activity.In addition to the matrix destroying effect, certain of these MMPs suchas MMP-3 have been implemented as the in vivo activator for other MMPssuch as MMP-1 and MMP-9 (Ito, et al., Arch Biochem Biophys. 267, 211(1988); Ogata, et al., J. Biol. Chem. 267, 3581 (1992)). Thus, a cascadeof proteolytic activity can be initiated by an excess of MMP-3. Itfollows that specific MMP-3 inhibitors should limit the activity ofother MMPs that are not directly inhibited by such inhibitors.

It has also been reported that MMP-3 can cleave and thereby inactivatethe endogenous inhibitors of other proteinases such as elastase(Winyard, et al., FEBS Letts. 279 1, 91 (1991)). Inhibitors of MMP-3could thus influence the activity of other destructive proteinases bymodifying the level of their endogenous inhibitors.

A number of diseases are thought to be mediated by excess or undesiredmatrix-destroying metalloprotease activity or by an imbalance in theratio of the MMPs to the TlMPs. These include: a) osteoarthritis(Woessner, et al., J. Biol. Chem., 259(6), 3633 (1984); Phadke, et al.,3. Rheumatol. 10, 852 1983)), b) rheumatoid arthritis (Mullins, et al.,Biochim. Biophys. Acta 695, 117 (1983)); Woolley, et al., ArthritisRheum. 20, 1231 (1977); Gravallese, et al., Arthritis Rheum. 34, 1076(1991)), c) septic arthritis (Williams, et al., Arthritis Rheum. 33, 533(1990)), d) tumor metastasis (Reich, et al., Cancer Res., 48, 3307(1988), and Matisian, et al., Proc. Nat'l. Acad. Sci., USA 83, 9413,(1986)), e) periodontal diseases (Overall, et al., J. Periodontal Res.22, 81 (1987)), f) corneal ulceration (Burns, et al., Invest. Opthalmol.Vis. Sci. 30, 1569 (1989)), g) proteinuria (Baricos, et al., Biochem. J.254, 609 (1988)), h) coronary thrombosis from atherosclerotic plaquerupture (Henney, et al., Proc. Nat'l. Acad. Sci., USA 88, 8154(1991)), 1) aneurysmal aortic disease (Vine, et al., Clin. Sci. 81, 233(1991)), j) birth control (Woessner, et al., Steroids 54, 491 (1989)),k) dystrophobic epidermolysis bullosa (Kronberger, et al., J. InvestDermatol. 79, 208 (1982)), and l) degenerative cartilage loss followingtraumatic joint injury, m) conditions leading to inflammatory responses,osteopenias mediated by MMP activity, n) tempero mandibular jointdisease, o) demyelating diseases of the nervous system (Chantry, et al.,J. Neurochem. 50, 688 (1988)).

The need for new therapies is especially important in the case ofarthritic diseases. The primary disabling effect of osteoarthritis (OA),rheumatoid arthritis (RA) and septic arthritis is the progressive lossof articular cartilage and thereby normal joint function. No marketedpharmaceutical agent is able to prevent or slow this cartilage loss,although nonsteroidal anti-inflammatory drugs (NSAIDs) have been givento control pain and swelling. The end result of these diseases is totalloss of joint function which is only treatable by joint replacementsurgery. MMP inhibitors are expected to halt or reverse the progressionof cartilage loss and obviate or delay surgical intervention.

Proteases are critical elements at several stages in the progression ofmetastatic cancer. In this process, the proteolytic degradation ofstructural protein in the basal membrane allows for expansion of a tumorin the primary site, evasion from this site as well as homing andinvasion in distant, secondary sites. Also, tumor induced angiogenesisis required for tumor growth and is dependent on proteolytic tissueremodeling. Transfection experiments with various types of proteaseshave shown that the matrix metalloproteases play a dominant role inthese processes in particular gelatinases A and B (MMP-2 and MMP-9,respectively). For an overview of this field see Mullins, et al.,Biochim. Biophys. Acta 695, 177 (1983); Ray, et al., Eur. Respir. J. 7,2062 (1994); Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. 4, 197(1993).

Furthermore, it was demonstrated that inhibition of degradation ofextracellular matrix by the native matrix metalloprotease inhibitorTIMP-2 (a protein) arrests cancer growth (DeClermk, et al., Cancer Res.52, 701 (1992)) and that TIMP-2 inhibits tumor-induced angiogenesis inexperimental systems (Moses, et al. Science 248, 1408 (1990)). For areview, see DeClerck, et al., Ann. N. Y. Acad. Sci. 732, 222 (1994). Itwas further demonstrated that the synthetic matrix metalloproteaseinhibitor batimastat when given intraperitoneally inhibits human colontumor growth and spread in an orthotopic model in nude mice (Wang, etal. Cancer Res. 54, 4726 (1994)) and prolongs the survival of micebearing human ovarian carcinoma xenografts (Davies, et. al., Cancer Res.53, 2087 (1993)). The use of this and related compounds has beendescribed in Brown, et al., WO-9321942 A2 (931111).

There are several patents and patent applications claiming the use ofmetalloproteinase inhibitors for the retardation of metastatic cancer,promoting tumor regression, inhibiting cancer cell proliferation,slowing or preventing cartilage loss associated with osteoarthritis orfor treatment of other diseases as noted above (e.g. Levy, et al.,WO-9519965 Al; Beckett, et al., WO-9519956 Al; Beckett, et al.,WO-9519957 Al; Beckett, etal., WO-9519961 Al; Brown, et al., WO-9321942A2; Crimmin, et al., WO-9421625 Al; Dickens, et al., U.S. Pat. No.4,599,361; Hughes, et al., U.S. Pat. No. 5,190,937; Broadhurst, et al.,EP 574758 Al; Broadhurst, et al,. EP 276436; and Myers, et al., EP520573 A1. The preferred compounds of these patents have peptidebackbones with a zinc complexing group (hydroxamic acid, thiol,carboxylic acid or phosphinic acid) at one end and a variety ofsidechains, both those found in the natural amino acids as well as thosewith more novel functional groups. Such small peptides are often poorlyabsorbed, exhibiting low oral bioavailability. They are also subject torapid proteolytic metabolism, thus having short half lives. As anexample, batimartat, the compound described in Brown, et al., WO-9321942A2, can only be given intra peritoneally.

Certain 3-biphenoylpropanoic and 4-biaryloylbutanoic acids are describedin the literature as anti-inflammatory, anti-platelet aggregation,anti-phlogistic, anti-proliferative, hypolipidemic, antirheumatic,analgesic, and hypocholesterolemic agents. In none of these examples isa reference made to MMP inhibition as a mechanism for the claimedtherapeutic effect. Certain related compounds are also used asintermediates in the preparation of liquid crystals.

Specifically, Tomcufcik, et al., U.S. Pat. No. 3,784,701 claims certainsubstituted benzoylpropionic acids to treat inflammation and pain. Thesecompounds include 3-biphenoylpropanoic acid (a.k.a fenbufen) shownbelow.

Child, et al., J. Pharm. Sci., 66, 466 (1977) describesstructure-activity relationships of several analogs of fenbufen. Theseinclude several compounds in which the biphenyl ring system issubstituted or the propanoic acid portion is substituted with phenyl,halogen, hydroxyl or methyl, or the carboxylic acid or carbonylfunctions are converted to a variety of derivatives. No compounds aredescribed which contain a 4′-substituted biphenyl and a substitutedpropanoic acid portion combined in one molecule. The phenyl (compoundsXLIX and LXXVII) and methyl (compound XLVII) substituted compounds shownbelow were described as inactive.

Kameo, et al., Chem. Pharm. Bull., 36, 2050 (1988) and Tomizawa, et al.,JP patent 62132825 A2 describe certain substituted 3-biphenoylpropionicacid derivatives and analogs thereof including the following. Variouscompounds with other substituents on the propionic acid portion aredescribed, but they do not contain biphenyl residues.

wherein X=H, 4′-Br, 4′-Cl, 4′-CH₃, or 2′-Br.

Cousse, et al., Eur. J. Med. Chem., 22, 45 (1987) describe the followingmethyl and methylene substituted 3biphenoyl-propanoic and -propenoicacids. The corresponding compounds in which the carbonyl is replacedwith either CH₂OH or CH₂ are also described.

wherein X=H, Cl, Br, CH₃O, F, or NH₂.

Nichl, et al. DE patent 1957750 also describes certain of the abovemethylene substituted biphenoylpropanoic acids.

El-Hashash, et al., Revue Roum. Chim. 23, 1581 (1978) describe productsderived from β-aroyl-acrylic acid epoxides including the followingbiphenyl compound. No compounds substituted on the biphenyl portion aredescribed.

Kitamura, et al., JP patent 60209539 describes certain biphenylcompounds used as intermediates for the production of liquid crystalsincluding the following. The biphenyl is not substituted in theseintermediates.

wherein R¹ is an alkyl of 1-10 carbons.

Thyes, et al., DE patent 2854475 uses the following compound as anintermediate. The biphenyl group is not substituted.

Sammour, et al., Egypt J. Chem. 15, 311 (1972) and Couquelet, et al.,Bull. Soc. Chim. Fr. 9, 3196 (1971) describe certain dialkylaminosubstituted biphenoylpropanoic acids including the following. In no caseis the biphenyl group substituted.

wherein R¹, R²=alkyl, benzyl, H, or, together with the nitrogen,morpholinyl.

Others have disclosed a series of biphenyl-containing carboxylic acids,illustrated by the compound shown below, which inhibit neuralendopeptidase (NEP 24.11), a membrane-bound zinc metalloprotease(Stanton, et al., Bioorg. Med. Chem. Lett. 4, 539 (1994); Lombaert, etal., Bioorg. Med. Chem. Lett. 4, 2715 (1994); Lombaert, et al., Bioorg.Med. Chem. Lett. 5, 145 (1995); Lombaert, et al., Bioorg. Med. Chem.Lett. 5, 151 (1995)).

It has been reported that N-carboxyalkyl derivatives containing abiphenylethylglycine, illustrated by the compound shown below, areinhibitors of stromelysin-1 (MMP-3), 72 kDA gelatinase (MMP-2) andcollagenase (Durette, et al., WO-9529689).

It would be desirable to have effective MMP inhibitors which possessimproved bioavailability and biological stability relative to thepeptide-based compounds of the prior art, and which can be optimized foruse against particular target MMPs. Such compounds are the subject ofthe present application.

The development of efficacious MMP inhibitors would afford new therapiesfor diseases mediated by the presence of, or an excess of MMP activity,including osteoarthritis, rheumatoid arthritis, septic aithritis, tumormetastasis, periodontal diseases, corneal ulcerations, and proteinuria.Several inhibitors of MMPs have been described in the literature,including thiols (Beszant, et al., J. Med. Chem. 36, 4030 (1993),hydroxamic acids (Wahl, et al. Bioorg. Med. Chem. Lett. 5, 349 (1995)Conway, et al. J. Exp. Med. 182, 449 (1995); Porter, et al., Bioorg.Med. Chem. Lett. 4, 2741 (1994); Tomczuk, et al., Bioorg. Med. Chem.Lett. 5, 343 (1995); Castelhano, et al., Bioorg. Med. Chem. Lett. 5,1415 (1995)), phosphorous-based acids (Bird, et al. J. Med. Chem. 37,158 (1994); Morphy, et al., Bioorg. Med. Chem. Lett. 4, 2747 (1994);Kortylewicz, et al., J. Med. Chem. 33, 263 (1990)), and carboxylic acids(Chapman, et al. J. Med. Chem. 36, 4293 (1993); Brown, et al. J. Med.Chem. 37, 674 (1994); Morphy, et al., Bioorg. Med. Chem. Lett. 4, 2747(1994); Stack, et al., Arch. Biochem. Biophys. 287, 240 (1991); Ye, etal., J. Med. Chem. 37, 206 (1994); Grobelny, et al., Biochemistry 24,6145 (1985); Mookhtiar, et al., Biochemistry 27, 4299 (1988)). However,these inhibitors generally contain peptidic backbones, and thus usuallyexhibit low oral bioactivity due to poor absorption and short half livesdue to rapid proteolysis. Therefore, there remains a need for improvedMMP inhibitors.

SUMMARY OF THE INVENTION

This invention provides compounds having matrix metalloproteaseinhibitory activity. These compounds are useful for inhibiting matrixmetalloproteases and, therefore, combating conditions to which MMP'scontribute. Accordingly, the present invention also providespharmaceutical compositions and methods for treating such conditions.

The compounds described relate to a method of beating a mammalcomprising administering to the mammal a matrix metalloproteaseinhibiting amount of a compound according to the invention sufficientto:

(a) alleviate the effects of osteoarthritis, rheumatoid arthritis,septic arthritis, periodontal disease, corneal ulceration, proteinuria,aneurysmal aortic disease, dystrophobic epidermolysis, bullosa,conditions leading to inflammatory responses, osteopenias mediated byMMP activity, tempero mandibular joint disease, demyelating diseases ofthe nervous system;

(b) retard tumor metastasis or degenerative cartilage loss followingtraumatic joint injury,

(c) reduce coronary thrombosis from athrosclerotic plaque rupture; or

(d) temporarily reduce fertility (i.e., act as effective birth controlagents).

The compounds of the present invention are also useful scientificresearch tools for studying functions and mechanisms of action of matrixmetalloproteases in both in vivo and in vitro systems. Because of theirMMP-inhibiting activity, the present compounds can be used to modulateMMP action, thereby allowing the researcher to observe the effects ofreduced MMP activity in the experimental biological system under study.

This invention relates to compounds having matrix metalloproteaseinhibitory activity and the generalized formula:

(T)_(x)A—B—D—E—G  (L)

In the above generalized formula (L), (T)_(x)A represents a substitutedor unsubstituted aromatic 6-membered ring or heteroaromatic 5-6 memberedring containing 1-2 atoms of N, O, or S. T represents one or moresubstituent groups, the subscript x represents the number of suchsubstituent groups, and A represents the aromatic or heteroaromaticring.

In the generalized formula (L), B represents an aromatic 6-membered ringor a heteroaromatic 5-6 membered ring containing 1-2 atoms independentlyselected from the group of N, O, or S. It is referred to as the B ringor B unit. When N is employed in conjunction with either S or O in the Bring, these heteroatoms are separated by at least one carbon atom.

In the generalized formula (L), D represents

In the generalized formula (L), E represents a chain of n carbon atomsbearing m substituents R⁶ in which the R⁶ groups are independentsubstituents, or constitute spiro or nonspiro rings. Rings may be formedin two ways: a) two groups R⁶are joined, and taken together with thechain atom(s) to which the two R⁶ group(s) are attached, and anyintervening chain atoms, constitute a 3-7 membered ring, or b) one groupR⁶ is joined to the chain on which this one group R⁶ resides, and takentogether with the chain atom(s) to which the R⁶ group is attached, andany intervening chain atoms, constitutes a 3-7 membered ring. The numbern of carbon atoms in the chain is 2 or 3, and the number m of R⁶substituents is an integer of 1-3. The number of carbons in the totalityof R⁶ groups is at least two.

Each group R⁶ is alkyl, alkenyl, alkynyl, heteroaryl, non-aromaticcyclic, and combinations thereof optionally substituted with one or moreheteroatoms. In the

In the generalized formula (L), E preferably represents a linear orcyclic alkyl moiety substituted with a mono- or bi-heterocyclic ringstructure.

In the generalized formula (L), G represents —PO₃H₂, —M

in which M represents —CO₂H, —CON(R¹¹)₂, wherein R¹¹ is H or samplealkyl, or —CO₂R¹², wherein R¹² is lower alkyl and R¹³ represents any ofthe side chains of the 19 noncyclic naturally occurring amino acids.

Pharmaceutically acceptable salts of these compounds are also within thescope of the invention.

In most related reference compounds of the prior art the biphenylportion of the molecule is unsubstituted, and the propanoic or butanoicacid portion is either unsubstituted or has a single methyl or phenylgroup. Presence of the larger phenyl group has been reported to causeprior art compounds to be inactive as anti-inflammatory analgesicagents. See, for example, Child, et al., J. Pharm. Sci. 66, 466(1977).By contrast, it has now been found that compounds which exhibit potentMMP inhibitory activity contain a substituent of significant size on thepropanoic or butanoic portion of the molecule. The biphenyl portions ofthe best MMP inhibitors also preferably contain a substituent on the4′-position, although when the propanoic or butanoic portions areoptimally substituted, the unsubstituted biphenyl compounds of theinvention have sufficient activity to be considered realistic drugcandidates.

The foregoing merely summarizes certain aspects of the present inventionand is not intended, nor should it be construed, to limit the inventionin any way. All of the patents and other publications recited in thisspecification are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

More particularly, the compounds of the present invention are materialshaving matrix metalloprotease inhibitory activity and the generalizedformula:

(T)_(x)A—B—D—E—G  (L)

in which (T)_(x)A represents a substituted or unsubstituted aromatic orheteroaromatic moiety selected from the group consisting of:

in which R¹ represents H or alkyl of alkyl of 1-3 carbons.

Throughout this application, in the displayed chemical structures, anopen bond indicates the point at which the structure joins to anothergroup. For example,

where R⁵⁰ is

is the structure

In the above structures, for (T)_(x)A the aromatic ring is referred toas the A ring or A unit, and T represents a substituent group, referredto as a T group or T unit. Preferably 1.

The substituent group T can also be an acetylene containing moiety withthe general formula:

R³⁰(CH₂)_(n′)C=C

where n is 1-4 and R³⁰ is selected from the group consisting of: HO—,MeO—, N(n-Pr)₂—, CH₃CO₂—, CH₃CH₂OCO₂—, HO₂C—, OHC—, Ph—, 3—HO—Ph—, andPhCH₂O—, provided that when R³⁰ is Ph or 3—HO—Ph, n=0.

The B ring of generalized formula (L) is a substituted or unsubstitutedaromatic or heteroaromatic ring, in which any substituents are groupswhich do not cause the molecule to fail to fit the active site of thetarget enzyme, or disrupt the relative conformations of the A and Brings, such that they would be detrimental. Such substitutents may bemoieties such as lower alkyl, lower alkoxy, CN, NO₂, halogen, etc. butare not to be limited to such groups.

In the generalized formula (L), B represents an aromatic orheteroaromatic ring selected from the group consisting of:

in which R¹ is defined as above. These rings are referred to as the Bring or B unit.

In an alternative embodiment, compounds of the general formula (L)include those in which the combination (T)_(x)—A—B has the structure:

where Z may be (CH₂)_(c)—C₆H₄—(CH₂)_(f) or (CH₂)_(g), e=0-8, f=0-5 andg=0-14, r is 0-6. R¹⁵ may be a straight, or cyclic alkyl group of 6-12carbons atoms, preferably of 7-11 carbon atoms, and optionally may bearone or more pharmaceutically acceptable substituents which are discussedmore fully below.

R¹⁵ may also be a polyether of the formula R³²H(C₂H₄O)_(h) in which thesubscript “h” is 1 or 2, and the group R³² is a straight, branched orcyclic alkyl group of 1-5 carbon atoms, preferably of 1-3 carbon atomsand straight, or phenyl, or benyl. R³² optionally may bear one or morepharmaceutically-acceptable substituents.

R¹⁵ may also be a substituted alkynyl group of the formula:

R³³(CH₂)_(b)—C=C—

in which the subscript “b” is 1-10 and the group R³³ is H—, HO— or R³⁴O—and the group is preferably the HO— group. R³⁴ may be an alkyl group of1-3 carbon atoms, or phenyl or benzyl. R³³ optionally may bear one ormore pharmaceutically-acceptable substituents.

R¹⁵ may also be H, Cl, MeO or

wherein n is 0-4, R¹⁷ is C₂H₅, allyl, or benzyl.

In the generalized formula (L), D represents the moieties:

In the generalized formula (L), the moiety between D and G shown by thefollowing formula:

wherein r is 0-2 and R⁴⁰ is a mono- or bi- heterocyclic structure. Whenr=0 the above structure takes the form

When r is 1 or 2, a cyclobutyl or cyclopentyl ring is formed,respectively. Each ring of the mono- or bi- heterocylic structurescomprise 5-7 membered rings substituted with 1-3 heteroatomsindependently selected from N, S, and O; one or two carbons of the ringare optionally carbonyl carbons; any sulfur of the ring is optionally—S(O)— or —S(O)₂—; one or more ring members are optionally substitutedwith one or two methyl groups,

In addition, aryl or heteroaryl portions of any of the T or R⁶ groupsoptionally may bear up to two such as —(CH₂)_(y)C(R¹¹)(R¹²)OH,—(CH₂)_(y)OR¹¹, —(CH₂)_(y)SR¹¹, —CH₂)_(y)S(O)R¹¹, —CH₂)_(y)S(O)₂R¹¹,—CH₂)_(y)SO₂N(R¹¹)₂, —(CH₂)_(y)N(R¹¹)₂, —(CH₂)_(y)N(R¹¹)COR¹²,—OC(R¹¹)₂O— in which both oxygen atoms are connected to the aryl ring.

The B ring is preferably a 1,4-phenylene or 2,5-thiophene ring, mostpreferably 1,4-phenylene.

The D unit is most preferably a carbonyl group.

In the E unit, r is preferably 0 or 2 and R⁴⁰ is preferably one of thefollowing:

or PhCH₂OCH₂OCH_(2—).

The G unit is most preferably a carboxylic acid group and is attached tothe E unit at the 2 position, i.e., the carbon atom of the E unit betato the D unit.

It is to be understood that as used herein, the term “alkyl” meansstraight, branched, cyclic, and polycyclic materials. The term“haloalkyl” means partially or fully halogenated alkyl groups such as—(CH₂)₂Cl, —CF₃ and —C₆F₁₃ for example.

In the generalized formula (L), the A and B rings are preferably phenyland phenylene, respectively, the A ring preferably bears at least onesubstituent group T preferably located on the position furthest from theposition of the A ring which is connected to the B ring, the D unit ispreferably a carbonyl group, and the G unit is preferably a carboxylgroup.

Certain alternative embodiments include compounds having matrixmetalloproteinase inhibitory activity and the following generalizedformula:

where Z=(CH₂)_(c)—C₆H₄—(CH₂)_(g) or (CH₂)_(g), e=0-8, f=0-5, g=0-14, ris 0-6 and where y is 0, 2, or 3.

R¹⁵ may be H, Cl, MeO or

wherein n is 0-4, R¹⁷ is C₂H₅, allyl or benzyl, and R⁴⁰ is one of:

and —CH₂OCH₂OCH₂Ph.

The most preferred compounds of generalized formula (L) are

wherein T is selected from a group consisting of:

r is 0-2, and R⁴⁰ is selected from the group consisting of:

and —CH₂OCH₂OCH₂Ph.

The invention also relates to certain intermediates useful in thesynthesis of some of the claimed inhibitors. These intermediates arecompounds having the generalized formula

where Bn is benzyl, TMSE is trimethylsilyl ethyl and R⁴⁰ is as definedabove.

Those skilled in the art will appreciate that many of the compounds ofthe invention exist in enantiomeric or diastereomeric forms, and that itis understood by the art that such stereoisomers generally exhibitdifferent activities in biological systems. This invention encompassesall possible stereoisomers which possess inhibitory activity against anMMP, regardless of their stereoisomeric designations, as well asmixtures of stereoisomers in which at least one member possessesinhibitory activity.

The most prefered compounds of the present invention are as indicatedand named in the list below:

I)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl]-cyclopentanecarboxylicacid,

II)2-[(4′-chloro[1,1′-biphenyl)-4-yl)carbonyl]-5-[phenoxymethoxymethyl]-cyclopentanecarboxylicacid,

III)2-[4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[[(1-pyrrolidinylthioxomethyl)thio]methyl]-cyclopentanecarboxylicacid,

IV)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl]-cyclopentanecarboxylicacid,

V)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[1-oxo-2(1H)-phthalazinyl)methyl]-cyclopentanecarboxylicacid,

VI) 2-[4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(2-oxo-3(2H)-benzoxazolyl)methyl]-cyclopentanecarboxylicacid,

VII)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[5,5-dimethyl-2,4-dioxo-3-oxazolidinylmethyl]-cyclopentanecarboxylicacid,

VIII)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(2,4-dioxo-3-thiazolidinyl)methyl]-cyclopentanecarboxylicacid,

IX)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[2,4,5-trioxo-1-imidazolidinyl)methyl]-cyclopentanecarboxylacid

X)2-[(4′-chloro(1,1′-biphenyl]-4-yl)carbonyl]-5-[(3,6-dihydro-2,6-dioxo-1(2H)-pyrimidinyl)methyl]-cyclopentanecarboxylicacid,

XI)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinyl)methyl]-cyclopentanecarboxylicacid,

XII)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(1,4-dihydro-2,4-dioxo-3(2H)-quinazolinyl)methyl]-cyclopentanecarboxylicacid,

XIII)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[3,4-dihydro-1,3-dioxo-2(1H)-isoquinolinyl)methyl]-cyclopentanecarboxylicacid,

XIV)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(1,4-dihydro-4-oxo-3(2H)-quinazolinyl)methyl]-cyclopentanecarboxylicacid,

XV)2-[4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(1,3-dihydro-3-oxo-2H-indazol-2-yl)methyl]-cyclopentanecarboxylicacid,

XVI)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[2,3-dihydro-1H-benzimidazol-1-yl)methyl]-cyclopentanecarboxylicacid,

XVII)2-[(4′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(3,4-dihydro-1,4-dioxo-2(1H)-phthalazinyl)methyl]-cyclopentanecarboxylicacid,

XVIII) R/Sα-[2-(4′-chloro[1,1′-biphenyl]-4yl)2-oxoethyl]-1-oxo-2(1H)-phthalazinebutanoicacid,

XIX)R-α-[2-(4′-chloro[1,1′-biphenyl]-4-yl)-2-oxoethyl]-1-oxo-2(1H)-phthalazinebutanoicacid,

XX) S-α-[2-(4′-chloro[1,1′-biphenyl]-4-yl)-2-oxoethyl]-1-oxo-2(1H)-phthalazinebutanoic acid,

XXI)α-[2-(4′-chloro[1,1′-biphenyl]-4-yl)-2-oxoethyl]-4-oxo-1,2,3,-Benzotriazine-3(4H)-butanoicacid, and

XXII)α-[2-(4′-chloro[1,1′-biphenyl]-4-yl)-2-oxoethyl]-2,3-dihydro-5-methyl-2-oxo-1H-1,4-benzodiazepine-1-butanoicacid.

General Preparative Methods

The compounds of the invention may be prepared readily by use of knownchemical Ad0 reactions and procedures. Nevertheless, the followinggeneral preparative methods are presented to aid the reader insynthesizing the inhibitors, with more detailed particular examplesbeing presented below in the experimental section describing the workingexamples. AU variable groups of these methods are as described in thegeneric description if they are not specifically defined below. Thevariable subscript n is independently defined for each method. When avariable group with a given symbol (i.e R⁹) is used more than once in agiven structure, it is to be understood that each of these groups may beindependently varied within the range of definitions for that symbol.

General Method A—The compounds of this invention in which the rings Aand B are substituted phenyl and phenylene respectively are convenientlyprepared by use of a Friedel-Crafts reaction of a substituted biphenylMII with an activated acyl-containing intermediate such as the succinicor glutaric anhydride derivative MIII or acid chloride MIV in thepresence of a Lewis acid catalyst such as aluminum trichloride in anaprotic solvent such as 1,1,2,2-tetrachloroethane. The well knownFriedel-Crafts reaction can be accomplished with use of many alternativesolvents and acid catalysts as described by Berliner, Org. React, 5,229, 1949 and Heaney, Comp. Org. Synth. 2, 733, 1991.

If the anhydride MIII is monosubstituted or multiply-substituted in anunsymmetrical way, the raw product MI-A often exists as a mixture ofisomers via attack of the anhydride from either of the two carbonyls.The resultant isomers can be separated into pure forms bycrystallization or chromatography using standard methods known to thoseskilled in the art.

When they are not commercially available, the succinic anhydrides MIIIcan be prepared via a Stobbe Condensation of a dialkyl succinate with analdehyde or ketone (resulting in side chain R⁶), followed by catalytichydrogenation, hydrolysis of a hemiester intermediate to a diacid, andthen conversion to the anhydride MIII by reaction with acetyl chlorideor acetic anhydride. Alternatively, the hemiester intermediate isconverted by treatment with thionyl chloride or oxalyl chloride to theacid chloride MIV. For a review of the Stobbe condensation, includinglists of suitable solvents and bases see Johnson and Daub, Org. React.,6, 1, 1951.

This method, as applied to the preparation of MIII (R⁶=H, isobutyl andH, n-pentyl), has been described Wolanin, et al., U.S. Pat. No.4,771,038.

Method A is especially useful for the preparation of cyclic compoundssuch as MI-A-3, in which two R⁶ groups are connected in a methylenechain to form a 3-7 member ring. Small ring (3-5 member) anhydrides arereadily available only as cis isomers which yield cis inventioncompounds MI-A-3. The trans compounds Ml-A4 are then prepared bytreatment of MI-A-3 with a base such as DBU in THF. The substituted fourmember ring starting material anhydrides such as MIII-A-1 are formed ina photochemical 2+2 reaction as shown below. This method is especiallyuseful for the preparation of compounds in which R¹⁴ is acetoxy oracetoxymethylene. After the subsequent Friedel-Crafts reaction theacetate can be removed by basic hydrolysis and the carboxyl protected byconversion to 2-(trimethylsilylethyl ester. The resultant intermediatewith R¹⁴=CH₂OH can be converted to invention compounds with other R¹⁴groups by using procedures described in General Method G.

The Friedel-Crafis method is also usefu when double bonds are foundeither between C-2 and C-3 of a succinoyl chain (from maleic anhydrideor 1-cyclopentene-1,2-dicarboxylic anhydride, for example) or when adouble bond is found in a side chain, such as in the use of itaconicanhydride as starting material to yield products in which two R⁶ groupsare found on one chain carbon together to form an exo-methylene (=CH₂)group. Subsequent uses of these compounds are described in Methods D.

General Method B—Alternatively the compounds MI can be prepared via areaction sequence involving mono-alkylation of a dialkyl malonate MVIwith an alkyl halide to form intermediate MVII, followed by alkylationwith a halomethyl biphenyl ketone MVIII to yield intermediate MIX.Compounds of structure MIX are then hydrolyzed with aqueous base andheated to decarboxylate the malonic acid intermediate and yield MI-B-2(Method B-1). By using one equivalent of aqueous base the esters MN-B-2with R¹² as alkyl are obtained, and using more than two equivalents ofbase the acid compounds (R¹²=H) are obtained. Optionally, heat is notused and the diacid or acid-ester MI-B-1 is obtained.

Alternatively, the diester intermediate MIX can be heated with a strongacids such as concentrated hydrochloric acid in acetic acid in a sealedtube at about 110° C. for about 24 hr to yield MI-B-1 (R¹²=H).Alternatively, the reaction of MVI with MVIII can be conducted beforethat with the alkyl halide to yield the same MIX (Method B-2).

Alternatively, a diester intermediate MIX which contains R¹²=allyl, canbe exposed to Pd catalysts in the presence of pyrrolidine to yieldMI-B-2 (R¹²=H) (Dezeil, Tetrahedron Lett. 28, 4371, 1990.

Intermediates MVII are formed from biphenyls MII in a Friedel-Craftreaction with haloacetyl halides such as bromoacetyl bromide orchloroacetyl chloride. Alternatively, the biphenyl can be reacted withacetyl chloride or acetic anhydride and the resultant producthalogenated with, for example, bromine to yield intermediates MVI(X=Br).

Method B has the advantage of yielding single regio isomers when MethodA yields mixtures. Method B is especially useful when the side chains R⁶contain aromatic or heteroaromatic rings that may participate inintramolecular acylation reactions to give side products if Method Awere to be used. This method is also very useful when the R⁶ groupadjacent to the carboxyl of the final compound contains heteroatoms suchas oxygen, sulfur, or nitrogen, or more complex functions such as imiderings.

When R⁶ contains selected functional groups Z, malonate MVII can beprepared by alkylating a commercially available unsubstituted malonatewith prenyl or allyl halide, subject this product to ozonalysis withreductive work-up, and the desired z group can be coupled via aMitsunobu reaction (Mitsunobu, Synthesis 1, 1981). Alternatively, theintermediate alcohol can be subjected to alkylation conditions toprovide malonate MVII containing the desired Z group.

General Method C—Especially useful is the use of chiral HPLC to separatethe enantiomers of racemic product mixtures (see, for example, Arit, etal., Chem. Int Ed. Engl. 12, 30 (1991)). The compounds of this inventioncan be prepared as pure enantiomers by use of a chiral auxiliary route.See, for example, Evans, Aldrichimica Acta, 15(2), 23, 1982 and othersimilar references known to one skilled in the art.

General Method D—Compounds in which R⁶ are alkyl- or aryl- orheteroaryl- or acyl- or a heteroarylcarbonyl-thiomethylene are preparedby methods analogous to those described in the patent WO 90/05719. Thussubstituted itaconic anhydride MXVI (n=1) is reacted underFriedel-Crafts conditions to yield acid MI-D-1 which can be separated bychromatography or crystallization from small amounts of isomeric MI-D-5.Alternatively, MI-D-5s are obtained by reaction of invention compoundsMI-D-4 (from any of Methods A through C) with formaldehyde in thepresence of base.

Compounds MI-D-1 or MI-D-5 are then reacted with a mercapto derivativeMXVII or MXVIII in the presence of catalyst such as potassium carbonate,ethyldiisobutylamine, tetrabutylammonium fluoride or free radicalinitiators such as azobisisobutyronitile (AIBN) in a solvent such asdiethylformamide or tetrahydrofuran to yield invention compounds MI-D-2,MI-D-3, MI-D-6, or MI-D-7.

General Method E—Biaryl compounds such as those of this application mayalso be prepared by Suzuki or Stille cross-coupling reactions of aryl orheteroaryl metallic compounds in which the metal is zinc, tin,magnesium, lithium, boron, silicon, copper, cadmium or the like with anaryl or heteroaryl halide or triflate (trifluoromethane-sulfonate) orthe like. In the equation below either Met or X is the metal and theother is the halide or triflate (OTf). Pd(com) is a soluble complex ofpalladium such as tetrakis(triphenylphosphine)palladium(O) or bis-(triphenylphosphine)palladium(E) chloride. These methods are well knownto those skilled in the art. See, for example, Suzuki, Pure Appl. Chem.63, 213 (1994); Suzuki, Pure Appl. Chem. 63, 419 (1991); and Farina andRoth, “Metal-Organic Chemistry” Volume 5 (Chapter 1), 1994.

The starting materials MXXIII (B=1,4-phenylene) are readily formed usingmethods analogous to those of methods A, B, C, or D but using ahalobenzene rather Man a biphenyl as staring material. When desired, thematerials in which X is halo can be converted to those in which X ismetal by reactions well known to those skilled in the art, such astreatment of a bromo intermediate with hexamethylditin and palladiumtetrakistriphenylphosphine in toluene at reflux to yield thetrimethyltin intermediate. The starting materials MXXIII (B=heteroaryl)are most conveniently prepared by method C but using readily availableheteroaryl rather than biphenyl starting materials. The intermediatesMXXII are either commercial or easily prepared from commercial materialsby methods well known to those skilled in the art.

These general methods are useful for the preparation of compounds forwhich Friedel-Crafts reactions such as those of Methods A, B, C, or Dwould lead to mixtures with various biaryl acylation patterns. Method Eis also especially useful for the preparation of products in which thearyl groups, A or B, contain one or more heteroatoms (heteroaryls) suchas those compounds that contain thiophene, furan, pyridine, pyrrole,oxazole, thiazole, pyrimidine or pyrazine rings or the like instead ofphenyls.

General Method F—When the R⁶ groups of method F form together a 4-7member carbocyclic ring as in Intermediate MXXV below, the double bondcan be moved out of conjugation with the ketone group by treatment withtwo equivalents of a strong base such as lithium diisopropylamide orlithium hexamethylsilylamide or the like followed by acid quench toyield compounds with the structure MXVI. Reaction of MXXVI with mercaptoderivatives using methods analogous to those of General Method D thenleads to cyclic compounds MI-F-I or MI-F-2.

General Method G—The compounds of this invention in which two R⁶ groupsare joined to form a substituted 5-member ring are most convenientlyprepared by method G. In this method acid CLII (R=H) is prepared usingthe protocols described in Tetrahedron 37, Suppl., 411 (1981). The acidis protected as an ester [eg. R=benzyl (Bn) or 2-(trimethylsilyl)ethyl(TMSE)] by use of coupling agents such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride andprocedures well known to those skilled in the art. Substitutedbromobiphenyl CIII is converted to its Grignard reagent by treatmentwith magnesium and reacted with CLII to yield alcohol CVI. Alcohol CVIis eliminated via base treatment of its mesylate by using conditionswell known to those skilled in the art to yield olefin CVII.Alternatively CIII is converted to a trimethyltin intermediate viainitial metallation of the bromide with n-butyllithium at lowtemperature (−78° C.) followed by treatment with chlorotrimethyltin andCI is converted to an enoltriflate (CII) by reaction with2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chloropyridine in thepresence of a strong aprotic base. The tin and enoltrifiateintermediates are then coupled in the presence of a Pd⁰ catalyst, CuIand AsPh₃ to yield directly intermediate CVII. Ozonolysis of CVII(workup with methylsufide) yields aldehyde CVIII. Alternativelytreatment with OsO₄ followed by HIO₄ converts CVII to CVIII.

Conversion of key intermediate CVIII to the targeted patent compounds isaccomplished in several ways depending on the identity of side chainfunction Z. Reaction of CVIII with Wittig reagents followed byhydrogenation yields products in which Z is alkyl and or arylalkyl.Selective reduction of aldehyde CVIII with a reducing agent such aslithium tris [(3-ethyl-3pentyl)oxy]aluminum hydride (LTEPA) yieldsalcohol CIX. The alcohol is converted to phenyl ethers or a variety ofheteroatom substituted derivatives used to generate sidechain Z via theMitsunobu reaction using conditions well known to those skilled in theart (see Mitsunobu, Synthesis, 1 (1981)). Alternatively the alcohol ofCIX is converted to a leaving group such as tosylate (CX) or bromide byconditions well known to those skilled in the art and then the leavingLa group is displaced by an appropriate nucleophile. Several examples ofthis type of reaction can be found in Norman, et al., J. Med. Chem. 37,2552 (1994). Direct acylation of the alcohol CIX yields inventioncompounds in which Z=OAcyl and reaction of the alcohol with variousallcyl halides in the presence of base yields alkyl ethers. In each casea final step is removal of acid blocking group R to yield acids (R=H) byusing conditions which depend on the stability of R and Z, but in allcases well known to those skilled in the art such as removal of benzylby base hydrolysis or of 2-(trimethylsilyl)ethyl by treatment withtetrabutylammonium fluoride.

General Method H—Amides of the acids of the invention compounds can beprepared from the acids by treatment in an appropriate solvent such asdichloromethane or dimethylformamide with a primary or secondary amineand a coupling agent such as dicyclohexylcarbodiimide. These reactionsare well known to those skilled in the art. The amine component can besimple alkyl or arylalkyl substituted or can be amino acid derivativesin which the carboxyl is blocked and the amino group is free.

General Method I—The compounds of this invention in which (T)_(x) is analkynyl or substituted alkynyl are prepared according to general methodI (Austin, J. Org. Chem. 46, 2280 (1981)). Intermediate MX is preparedaccording to methods A, B, C, D or G by starting with commercial MII(T=Br). Reaction of MX with substituted acetylene MXI in the presence ofCu(I)/palladate reagent gives invention compound MI-I-1. In certaincases, R³ may be an alcohol blocked as trialkylsilyl. In such cases thesilyl group can be removed by treatment with acids such astrifluoroacetic acid or HF - pyridine reagent.

Suitable pharmaceutically acceptable salts of the compounds of thepresent invention include addition salts formed with organic orinorganic bases. The salt forming ion derived from such bases can bemetal ions, e.g., aluminum, alkali metal ions, such as sodium ofpotassium, alkaline earth metal ions such as calcium or magnesium, or anamine salt ion, of which a number are known for this purpose. Examplesinclude ammonium salts, arylalkylamines such as dibenzylamnine andN,N-dibenzylethylenediamine, lower alkylamines such as methylamine,t-butylarine, procaine, lower alkylpiperidines such asN-ethylpiperidine, cycloalkylamines such as cyclohexylamine ordicyclohexylamine, 1-adamantylamine, benzathine, or salts derived fromamino acids like arginine, lysine or the like. The physiologicallyacceptable salts such as the sodium or potassium salts and the aminoacid salts can be used medicinally as described below and are preferred.

These and other salts which are not necessarily physiologicallyacceptable are useful in isolating or purifying a product acceptable forthe purposes described below. For example, the use of commerciallyavailable enantiomerically pure amines such as (+)-cinchonine insuitable solvents can yield salt crystals of a single enatiomer of theinvention compounds, leaving the opposite enantiomer in solution in aprocess often referred to as “classical resolution.” As one enantiomerof a given invention compound is usually substantially greater inphysiological effect than its antipode, this active isomer can thus befound purified in either the crystals or the liquid phase. The salts areproduced by reacting the acid form of the invention compound with anequivalent of the base supplying the desired basic ion in a medium inwhich the salt precipitates or in aqueous medium and then lyophilizing.The free acid form can be obtained from the salt by conventionalneutralization techniques, e.g., with potassium bisulfate, hydrochloricacid, etc.

The compounds of the present invention have been found to inhibit thematrix metalloproteases MMP-3, MMP-9 and MMP-2, and to a lesser extentMMP-1, and are therefore useful for treating or preventing theconditions referred to in the background section. As other MMPs notlisted above share a high degree of homology with those listed above,especially in the catalytic site, it is deemed that compounds of theinvention should also inhibit such other MMPs to varying degrees.Varying the substituents on the biaryl portions of the molecules, aswell as those of the propanoic or butanoic acid chains of the claimedcompounds, has been demonstrated to affect the relative inhibition ofthe listed MMPs. Thus compounds of this general class can be “tuned” byselecting specific substituents such that inhibition of specific MMP(s)associated with specific pathological conditions can be enhanced whileleaving non-involved MMPs less affected.

The method of treating matrix metalloprotease-mediated conditions may bepracticed in mammals, including humans, which exhibit such conditions.

The inhibitors of the present invention are contemplated for use inveterinary and human applications. For such purposes, they will beemployed in pharmaceutical compositions containing active ingredient(s)plus one or more pharmaceutically acceptable carriers, diluents,fillers, binders, and other excipients, depending on the administrationmode and dosage form contemplated.

Administration of the inhibitors may be by any suitable mode known tothose skilled in the art Examples of suitable parenteral administrationinclude intravenous, intraarticular, subcutaneous and intramuscularroutes. Intravenous administration can be used to obtain acuteregulation of peak plasma concentrations of the drug. Improved half-lifeand targeting of the drug to the joint cavities may be aided byentrapment of the drug in liposomes. It may be possible to improve theselectivity of liposomal targeting to the joint cavities byincorporation of ligands into the outside of the liposomes that bind tosynovial-specific macromolecules. Alternatively intramuscular,intraarticular or subcutaneous depot injection with or withoutencapsulation of the drug into degradable microspheres e.g., comprisingpoly(DL-lactide-co-glycolide) may be used to obtain prolonged sustaineddrug release. For improved convenience of the dosage form it may bepossible to use an i.p. implanted reservoir and septum such as thePercuseal system available from Pharmacia Improved convenience andpatient compliance may also be achieved by the use of either injectorpens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g.from Bioject, Mediject or Becton Dickinson). Prolonged zero-order orother precisely controlled release such as pulsatile release can also beachieved as needed using implantable pumps with delivery of the drugthrough a cannula into the synovial spaces. Examples include thesubcutaneously implanted osmotic pumps available from ALZA, such as theALZET osmotic pump.

Nasal delivery may be achieved by incorporation of the drug intobioadhesive particulate carriers (<200 μm) such as those comprisingcellulose, polyacrylate or polycarbophil, in conjunction with suitableabsorption enhancers such as phospholipids or acylcamitines. Availablesystems include those developed by DanBiosys and Scios Nova.

A noteworthy attribute of the compounds of the present invention incontrast to those of various peptidic compounds referenced in thebackground section of this application is the demonstrated oral activityof the present compounds. Certain compounds have shown oralbioavailability in various animal models of up to 90-98%. Oral deliverymay be achieved by incorporation of the drug into tablets, coatedtablets, dragees, hard and soft gelatine capsules, solutions, emulsionsor suspensions. Oral delivery may also be achieved by incorporation ofthe drug into enteric coated capsules designed to release the drug intothe colon where digestive protease activity is low. Examples include theOROS-CT/Osmet™ and PULSINCAP™ systems from ALZA and Scherer DrugDelivery Systems respectively. Other systems use azo-crosslinkedpolymers that are degraded by colon specific bacterial azoreductases, orpH sensitive polyacrylate polymers that are activated by the rise in pHat the colon. The above systems may be used in conjunction with a widerange of available absorption enhancers.

Rectal delivery may be achieved by incorporation of the drug intosuppositories.

The compounds of this invention can be manufactured into the abovelisted formulations by the addition of various therapeutically inertinorganic or organic carriers well known to those skilled in the art.Examples of these include, but are not limited to, lactose, corn starchor derivatives thereof, talc, vegetable oils, waxes, fats, polyols suchas polyethylene glycol, water, saccharose, alcohols, glycerin and thelike. Various preservatives, emulsifiers, dispersants, flavorants,wetting agents, antioxidants, sweeteners, colorants, stabilizers, salts,buffers and the like are also added, as required to assist in thestabilization of the formulation or to assist in increasingbioavailability of the active ingredient(s) or to yield a formulation ofacceptable flavor or odor in the case of oral dosing.

The amount of the pharmaceutical composition to be employed will dependon the recipient and the condition being treated. The requisite amountmay be determined without undue experimentation by protocols known tothose skilled in the art. Alternatively, the requisite amount may becalculated, based on a determination of the amount of target enzymewhich must be inhibited in order to treat the condition.

The matrix metalloprotease inhibitors of the invention are useful notonly for treatment of the physiological conditions discussed above, butare also useful in such activities as purification of metalloproteasesand testing for matrix metalloprotease activity. Such activity testingcan be both in vitro using natural or synthetic enzyme preparations orin vivo using, for example, animal models in which abnormal destructiveenzyme levels are found spontaneously (use of genetically mutated ortransgenic animals) or are induced by administration of exogenous agentsor by surgery which disrupts joint stability.

EXPERIMENTAL

General Procedures

All reactions were performed in flame-dried or oven-died glassware undera positive pressure of argon and were stirred magnetically unlessotherwise indicated. Sensitive liquids and solutions were transferredvia syringe or cannula and were introduced into reaction vessels throughrubber septa. Reaction product solutions were concentrated using a Buchievaporator unless otherwise indicated.

Materials

Commercial grade reagents and solvents were used without furtherpurification except that diethyl ether and tetrahydrofuran were usuallydistilled under argon from benzophenone ketyl, and methylene chloridewas distilled under argon from calcium hydride. Many of the specialtyorganic or organometallic starting materials and reagents were obtainedfrom Aldrich, 1001 West Saint Paul Avenue, Milwaukee, Wis. 53233.Solvents are often obtained from EM Science as distributed by VWRScientific.

Chromatography

Analytical thin-layer chromatography (MC) was performed on Whatran®pre-coated glass-backed silica gel 60 A F-254 250 μm plates.Visualization of spots was effected by one of the following techniques:(a) ultraviolet illumination, (b) exposure to iodine vapor, (c)immersion of the plate in a 10% solution of phosphomolybdic acid inethanol followed by heating, and (d) immersion of the plate in a 3%solution of p-anisaldehyde in ethanol containing 0.5% concentratedsulfuric acid followed by heating, and e) immersion of the plate in a 5%solution of potassium permanganate in water containing 5% sodiumcarbonate followed by heating.

Column chromatography was performed using 230-400 mesh EM Science®silica gel.

Analytical high performance liquid chromatography (HPLC) was performedat 1 mL min⁻¹ on a 4.6×250 mm Microsorb® column monitored at 288 nm, andsemi-preparative HPLC was performed at 24 mL min⁻¹ on a 21.4×250 mmMicrosorb® column monitored at 288 nm.

Instrumentation

Melting points (mp) were determined with a Thomas-Hoover melting pointapparatus and are uncorrected.

Proton (¹H) nuclear magnetic resonance (NMR) spectra were measured witha General Electric GN-OMEGA 300 (300 MHz) spectrometer, and carbonthirteen (¹³C) NMR spectra were X measured with a General ElectricGN-OMEGA 300 (75 MHz) spectrometer. Most of the compounds synthesized inthe experiments below were analyzed by NMR, and the spectra wereconsistent with the proposed structures in each case.

Mass spectral (MS) data were obtained on a Kratos Concept 1-Hspectrometer by liquid-cesium secondary ion (LCIMS), an updated versionof fast atom bombardment (FAB). Most of the compounds systhesized in theexperiments below were analyzed by mass spectroscopy, and the spectrawere consistent with the proposed structures in each case.

General Comments

For multi-step procedures, sequential steps are noted by numbers.Variations within steps are noted by letters. Dashed lines in tabulardata indicates point of attachment

EXAMPLE 1 Preparation of Compound I

Step 1

A solution of exo-oxobicyclo [2.2.1] heptane-7-carboxylic acid (preparedusing the protocols described in Tetrahedron, 37 suppl., 411, 1981)(3.04 g, 19.7 mmol) in CH₂Cl₂ (45 mL) was cooled to 0° C. and treatedwith 2-(trimethylsilyl) ethanol (2.7 mL, 18.6 mmol), EDC (3.94 g, 20.55mmol) and DMAP (0.11 g, 0.9 mmol). After warming to room temperature andstirring for 2 hrs., the reaction mixture was quenched with water anddiluted with CH₂Cl₂. After separating the layers, the organic phase waswashed with satd. aq. NaCl, dried over MgSO₄ and concentrated.Purification by MPLC (0-25% EtOAc-hexanes) provided the target compound(3.9 g, 78%) as a colorless oil. ¹H NMR (CDCl₃) δ4.18 (m, 2H), 2.88 (m,2H), 2.76 (m, 1H), 2.05 (m, 4H), 1.50 (m, 2H), 0.99 (t, J=8.4 Hz, 2H),0.99 (s, 9H).

Step 2

A solution of the ketone from step 1 (3.18 g, 12.50 mmol) and2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chloropyridine (6.6 g, 16.30mmol) in THF was cooled to −78° C. and carefully treated with a 0.5Msolution of KHMDS in toluene (24 mL, 12 mmol). After the addition wascomplete and the solution stirred for 2 h, the reaction mixture wasquenched with water (30 mL), warmed to room temperature and diluted withEtOAc. The two phases were the separated. The organic layer was washedwith satd. aq. NaCl, dried over MgSO₄ and concentrated. Purification byMPLC (0-15% EtOAc-hexanes) provided the target compound (4.2 g, 91%) asa colorless oil. ¹H NMR (CDCl₃) δ5.75 (d, J=4.8 Hz, 1H), 4.13 (t, J=9.0Hz, 2H), 3.18 (m, 2H), 2.62 (m, 1H), 2.62 (m, 2H), 1.41 (t, J=9.3 Hz,1H), 1.23 (t, J=9.1 Hz, 1H), 0.96 (t, J=8.4 Hz, 2H), 0.04 (s, 9H).

Step 3

A solution of 4chlorobiphenyl (3.0 g, 15.9 mmol) in acetic acid (50 mL)was carefully treated with bromine (1.1 mL, 20.7 mmol) at roomtemperature. The reaction mixture was heated to reflux for 4 h, cooledto room temperature and treated with excess propene until the mixturebecame clear. The solution was concentrated to a thick slurry, dilutedwith CH₂Cl₂ (50 mL) and washed successively with water and 2N NaOH. Theorganic extract was dried over MgSO₄, filtered and concentrated.Purification by re-crystallization form EtOAc gave the aryl bromide(3.57 g, 84%) as a white crystalline solid. ¹H NMR (CDCl₃) δ7.57 (m,2H), 7.48 (m, 2H), 7.41 (m, 4H).

Step 4

A solution of 4-bromo-4′-chlorobiphenyl (8.0 g, 30.0 mmol) in ThF (120mL) was cooled to −78° C. and carefully treated with n-BuLi (19.7 ml,1.6 M solution in hexanes, 31.5 mmol). After stirring for 1 h, themixture was treated with chlorotrimethyltin (33 mL, 1.0 M soln., 33.0mmol). After an additional 30 min., the solution was warned to roomtemperature and concnetrated. The off-white solid was diluted withCH₂Cl₂ (300 mL) and washed successively with water and satd. aq. NaCl.The organic layer was dried over MgSO₄, filtered and concentrated.Purification by MPLC (hexanes) gave the desired aryltin compound (9.38g, 89%) as a white crystalline solid. ¹H NMR (CDCl₃) δ7.62 (m, 6H), 7.54(m, 2H), 0.39 (s, 9H).

Step 5

A solution of the trrlate from step 2 (4.2 g, 10.89 mmol), CuI (0.215 g,1.1 mmol), AsPh₃ (0.339 g, 1.1 mmol), Cl₂Pd(MeCN)₂ (0.215 g, 0.56 mmol)and a few crystals of BHT in 1-methyl-2-pyrrolidinone (11.5 mL) waslowered into an oil bath preheated to 85° C. After stirring 4 min., thebiphenyltin derivative from step 4 (7.3 g, 20.7 mmol) was added in oneportion. The mixture was stirred for 30 min., cooled to room temperatureand diluted with EtOAc. After separating the phases, the aq. layer wasback extracted with EtOAc and the combined organic layers dried overMgSO₄, filtered and concentrated. The resulting residue was adsorbed onsilica gel and purified by MPLC (0-15% EtOAc-hexanes) to give thecoupled product (4.0 g, 86%) as a white crystalline solid. ¹H NMR(CDCl₃) δ7.52 (m, 6H), 7.42 (n, 2H), 6.40 (d, J=3.3 Hz, 1H), 4.19 (t,J=10.2 Hz, 2H), 3.58 (m, 1H), 3.23 (m, 1H), 2.60 (m, 1H), 1.95 (m, 2H),1.20 (m, 2H), 1.02 (d, J=7.5 Hz, 2H), 0.08 (s, 9H).

Step 6

A solution of the olefin from step 5 (3.60 g, 8.47 mmol) in 10%MeOH—CH₂Cl₂ (200 mL) was cooled to −78° C. and treated with ozone as agas added directly into the reaction mixture (10 min., 1 L/min.). AfterTLC indicated the absence of starting material the solution was purgedwith argon (15 min.), treated with methylsulfide (13 mL) and warmed toroom temperature. After stirring overnight, the solution wasconcentrated to a residue which was purified by MPLC (0-15%EtOAc-hexanes) to give a mixture of the desired aldehyde andcorresponding dimethyl acetal. The product mixture was dissolved inacetone (45 mL) and treated with CSA (0.192 g, 0.83 mmol) and water(0.13 mL, 16.5 mmol). After stirring overnight, the solution wasconcentrated and purified by MPLC (0-15% EtOAc-hexanes) to give thedesired aldehyde (3.45 g, 89%) as a colorless oil. NMR (CDCl₃) δ9.78 (d,J=9.0 Hz, 1H), 8.05 (d, J=6.6 Hz, 2H), 7.65 (d, J=6.6 Hz, 2H), 7.55 (d,J=9.0 Hz, 2H), 7.44 (d, J=9.0 Hz, 2H), 4.15 (m, 3H), 3.87 (t, J=7.2 Hz,1H), 3.15 (m, 1H), 2.20 (m, 1H), 2.03 (m, 1H), 1.86 (m, 1H), 1.58 (s,1H), 125 (t, J=6.9 Hz, 1H), 0.93 (m, 2H), 0.00 (s, 9H).

Step 7

A solution of lithium aluminum hydride (1.9 mL, 1.0 M THF) in THF (6 mL)was treated with 3-ethyl-3-pentanol (0.83 g, 5.77 mmol) and heated to agentle reflux for 1 h. The mixture was then cooled to room temperature.

A solution of the aldehyde intermediate from step 6 (0.85 g, 1.86 mmol)in THF (15 mL) was cooled to −78° C. and treated with the previouslyprepared solution of LTEPA in THF via cannula in a dropwise manner.After the addition was complete, the solution was stirred at −78° C. for4 h and subsequently quenched with 2N HCl (4.6 mL). The reaction mixturewas diluted with EtOAc and washed with water. The organic layer wasdried over MgSO₄, filtered and concnetrated. Purification by MPLC (5-40%EtOAc-hexanes) afforded the desired aldehyde (0.640 g, 75%) as a whitecrystalline solid. ¹H NMR (CDCl₃) δ8.05 (d, J=8.7 Hz, 2H), 7.65 (d,J=8.5 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 4.15 (m,2H), 3.76 (t, J=6.3 Hz, 2H), 3.28 (t, J=6.3 Hz, 2H), 2.48 (m, 1H), 2.35(t, J=6.0 Hz, 1H), 2.18 (m, 1H), 1.91 (m, 2H), 1.57 (s, 1H), 1.35 (t,J=6.9 Hz, 1H), 0.91 (m, 2H), −0.01 (s, 9H).

Step 8

A solution of the alcohol from step 7 (0.050 g, 0.109 mmol),triphenylphosphine (0.057 g, 0.217 mmol) andbenzo-1,2,3-triazin-4(3H)-one (0.034 g, 0.231 mmol) in THF (2.5 mL) wastreated with diethyl azodicarboxylate (0.035 mL, 0.222 mmol). Themixture was stirred at room temperature for 16 hrs., concentrated underreduced pressure and purified by MPLC (0-20% EtOAc-hexanes) to give thetarget compound (0.034 g, 53%). TLC: R_(f)0.16 (silica, 20%EtOAc-hexanes).

Step 9

A solution of the ester from step 8 (0.031 g, 0.052 mmol) in CH₂Cl₂ (2mL) was cooled to 0° C. and treated with TFA (0.25 mL). After stirringfor 5 h, the solution was concentrated under reduced pressure andpurified via flash column chromatography (0-5% MeOH-CH₂Cl₂) to give thedesired acid (0.023 g, 90%) as a white crystalline solid. MP 198-199° C.

EXAMPLE 2 Preparation of Compound II

Step 1

The benzyl ester was prepared in a manner analogous to the one describedfor the corresponding 2-trimethylsilyl ester intermediate (example 1,steps 1-7). In this case, benzyl alcohol was used instead of2-trimethylsilylethanol in step 1.

Step 2

A solution of the intermediate from step 1 (0.020 g, 0.045 mmol) anddiisopropylethylamine (0.025 mL, 0.144 mmol) in CH₂Cl₂ (1.5 mL) wastreated with benzyl chloromethylether (0.016 mL, 0.099 mmol) and stirredat room temperature for 6 h. Purification of the concentrated reactionmixture, by flash column chromatography (5-20% EtOAc-hexanes) providedthe desired ether (0.022 g, 86%). TLC:R_(f)0.25 (silica, 20%EtOAc-hexanes).

Step 3

A solution of the intermediate benzyl ester from step 2 (0.020 g, 0.035mmol) in ThF (0.4 mL) and ethanol (0.4mL was treated with NaOH solution(0.14 mL, 0.5 g/10 mL water). After stirring for 45 min At roomtemperature, the mixture was diluted with EtOAc and quenched with 2N HCl(0.2 ml). The organic layer was washed with satd. aq. NaCl, dried overMgSO₄ and concnetrated to give the desired acid (0.012 g, 72%). MP112-113° C.

EXAMPLE 3 Preparation of Compound III

Step 1

A solution of the alcohol from example 2, step 1 (0.100 g, 0.223 mmol)and diisopropylethylamine (0.05 mL, 0.287 mmol) in CH₂Cl₂ (3.0 mL) wastreated with p-toluenesulfonyl chloride (0.048 g, 0.249 mmol) and acrystal of DMAP. The mixture was stirred at room temperature for 16hrs.,concentrated under reduced pressure and purified by MPLC (0-20%EtOAc-hexanes) to give the desired tosylate (0.118 g, 88%).TLC:R_(f)0.23 (silica, 0-20% EtOAc-hexanes).

Step 2

A solution of the tosylate from step 1 (0.039 g, 0.066 mmol) and18-crown-6 (0.044 g, 0.166 mmol) in DMF (0.7 mL) was treated with sodiumpyrrolidine dithiocarbamate (0.035 g, 0.165 mmol) and stirred at roomtemperature for 16 h. The reaction mixture was diluted with EtOAc andwater. After separating the phases, the organic layer was washed withsatd. aq. NaCl, dried over MgSO₄, filtered and concentrated.Purification by MPLC (3-15% EtOAc-hexanes) provided the desired product(0.038 g, 99%). TLC:R_(f)0.34 (silica, 0-20% EtOAc-hexanes).

Step 3

The deprotection of the benzyl ester intermediate from step 2 wasaccomplished using the same protocol as described for example 2 in step3. MP 177-178° C.

The above methods for preparation of Examples 1-3 were, or could be usedto prepare the series of biphenyl containing products found in Table 1.

TABLE 1

Example R⁴⁰ Isomer Characterization I

R,S MP 198-199° C. II CH₂OCH₂OCH₂Ph R,S MP 112-130° C. III

R,S MP 177-178° C. IV

R,S R_(f) 0.33 (silica, 5% MeOH—CH₂Cl₂₎ V

R,S 219-220° C. VI

R,S 207° C. VII

R,S 210-211° C. VIII

R,S 290-291° C. IX

X

XI

XII

XIII

XIV

XV

XVI

XVII

EXAMPLE 18 Preparation of Compound XVIII

Step 1

A solution of pthlalazinone (1.00 g, 6.84 mmol), triphenylphosphine(1.79 g, 6.84 mmol) in THF (25 mL) was cooled to 0° C. and treated with2-bromo ethanol (0.480 mL, 6.84 mmol) and diethyl azocrboxylate (1.07mL, 6.84 mmol). After stirring 1 h at 0° C., the solution was warmed toroom temperature and stirred for an additional 12 h. The resultingmixture was concentrated and purified by flash column chromatography(35% ethyl acetate-hexanes) to afford 1.40 g (81%) of bromo ethylphthalazinone as a white solid. TLC:R_(f)0.65 (40% /ethylacetate-hexane).

Step 2

A solution of sodium hydride (0.040 g, 1.54 mmol) in THF (5 mL) wascooled to 0° C. and carefully treated with diallyl malonate (0.260 g,1.41 mmol). After warming to room temperature and stirring for 20 min.,bromo ethyl phthalazinone from step 1 (0.325 g, 1.28 mmol) was added inone portion and the mixture was heated to reflux for 18 h. The reactionmixture was diluted with saturated aq. NH₄Cl (20 mL) and EtOAc (20 mL).The resulting organic phase was washed with water, dried over MgSO₄,filtered, and concentrated to afford 0.240 g (52%) of a yellow oil.TLC:R_(f)0.60 (40% ethyl acetate-hexane).

Step 3

A 2L, three-necked, round bottom flask was equipped with a mechanicalstirrer, a thermometer and an argon inlet The flask was charged with asolution of 4-chlorobiphenyl (48.30 g, 0.256 mol) in dichloromethane(500 mL). Bromoacetyl bromide (23 mL, 0.26 mol) was added via syringeand the solution was cooled with an ice bath to an internal temperatureof 3° C. The thermometer was temporarily removed and AlCl₃ was addedportionwise over 5 min. The internal temperature rose to 10° C. andwhite gas evolved from the opaque olive green reaction mixture. After 24hrs. of stirring, the reaction was quenched by cautiously pouring intocold 10% HCl (1L). The organic layer became cloudy yellow green.Chloroform was added to help dissolve the solids, but the organic layernever became transparent. The organics were concentrated on a rotaryevaporator and dried further under vacuum. The crude product was a palegreen solid (˜82g) which was recrystallized from hot ethyl acetate togive 1-(2-bromoethanone)-4-(4-chlorophenyl)-benzene as brown needles(58.16 g). Concentration of the mother liquor followed by addition ofhexanes delivered a second crop of crystals (11.06 g) which gave an NMRspectrum identical to that of the first crop. The total yield of theproduct was 87%. TLC:R_(f)0.30 (silica, 70% hexanes-dichloromethane).

Step 4

A solution of sodium hydride (0.020 g, 0.775 mmol) in THF (2.0 mL) wascooled to 0° C. and carefully treated with the diester from step 2. Theice bath was removed and the resulting mixture was stirred for 20 min.The reaction mixture was re-cooled to 0° C. and treated with1-(2-bromoethanone)-4-(4-chlorophenyl)benzene (0.200 g, 0.646 mmol) inone portion. The mixture was warmed to room temperature over 30 min andsubsequently heated to reflux for 12 hrs. The reaction mixture was addedto satd. aq. NH₄Cl (10 mL) and diluted with EtOAc (10 mL). The resultingorganic phase was washed with water (10 mL), dried over MgSO₄, filteredand concentrated to afford 0.327 g (78%) of a yellow oil. TLC:R_(f)0.40(silica, 40% ethyl acetate-hexane).

Step 5

A solution of the diester product from step 4 (0.327 g, 0.558 mmol) in1,4 dioxane (5 mL) was treated with tetris(triphenylphosphine)palladium(0.006 g, 0.005 mmol) in one portion and pyrrolidone (0.102 mL, 1.22mmol) added dropwise over 15 min. After stirring for 30 min. at roomtemperature, the reaction mixture was diluted with 1N HCl (20 mL) andEtOAc (20 mL). The resulting organic phase was washed with satd. aq.NaCl, dried over MgSO₄, filtered, and concentrated to provide the diacidas a crude brown oil which was immediately carried on to step 6.TLC:R_(f)0.29 (silica, 5% methanol-methylene chloride).

Step 6

A solution of the diacid product from step 5 in 1,4 dioxane (25 mL) washeated to reflux for 24 h. After cooling to room temperature, theresulting mixture was concentrated to a gray solid. Recrystallizationfrom ethyl acetate afforded 0.044g (18%, two steps) of compound XVIII asa white solid. MP 232° C. TLC:R_(f)0.5 (silica, 10% methanol-methylenechloride).

EXAMPLE 19 Preparation of Compound XIX

Step 1

A solution of sodium hydride (0.040 g, 1.54 mmol) in THF (100 mL) wascooled to 0° C. and treated with di-tert-butyl malonate (20.7 3 mL,92.47 mmol) dropwise via dropping funnel, over 20 min. After stirring atroom temperature for 30 min., 3,3-dimethylallyl bromide (9.7 mL, 83.22mmol) was added. After siring an additional 19 h, the reaction mixturewas diluted with 10% HCl solution (100 mL) and EtOAc (100 mL). Theresulting organic phase was washed with satd. aq. NaCl, dried overMgSO₄, filtered, and concentrated to afford 25.74 g (94%)of a crudeyellow oil. TLC: R_(f)0.60 (silica, 10% ethyl acetate-hexane).

Step 2

A solution of the crude olefin from step 1 (25.74 g, 90.50 mmol) inCH₂Cl₂ (350 mL) and methanol (90 mL) was cooled to −78° C. and purgedwith 02 for 20 min. O₃ was bubbled through the solution until a bluecolor remained (2 h). The solution was purged with O₂ for 20 min.; untilthe solution became colorless. After warming to 0° C., NaBH₄ (3.42 g,90.50 mmol) was added in one portion. After several minutes the ice bathwas removed and the mixture was stirred overnight. The mixture wasconcentrated, reluted in CH₂Cl₂ , washed with water (100 mL), 10% HCl(100 mL), brine (50 mL), dried over MgSO₄, filtered and concentratedinto a colorless oil. Purification of 15.0 g of crude material by flashchromatography (30% ethyl acetate-hexanes) afforded 6.86 g (50%) as acolorless oil. TLC:R_(f)0.30 (silica, 35% ethyl acetate-hexane).

Step 3

The malonate intermediate was prepared in a manner analogous to the onedescribed for the preparation of example 18, step 1. For this example,benzo-1,2,3-triazin-4(3H)-one was used in place of phthalazinone and thealcohol form step 2 was used in place of 2-bromo ethanol. TLC:R_(f)0.40(silica, 40% ethyl acetate-hexane).

Step 4

The dialkylated malonate intermediate was prepared in a manner analogousto the one described for the preparation of example 18, step 2. In thisexample, the monoalkylated malonate from step 3 was alkylated with the1-(2-bromoethone)-4-(4-chlorophenyl)-benzene. TLC:R_(f)0.50 (silica, 40%ethyl acetate-hexane).

Step 5

A solution of the diester from step 4 (4.61 g, 0.746 mmol) in 1,4dioxane (10 mL) was treated with 4N HCl and heated to reflux for 10 h.After concnetrating to an oil, the residue was purified by flashchromatography (0-10% methanol-dichloromethane to give a yellow solid.MP 195° C.

EXAMPLE 20 AND EXAMPLE 21 Preparation of compounds XX and XXI

Example 19 was separated by chromatography on a chiral HPLC column(CH₂Cl₂EtOAc-hexanes). Example 20 was the first to come off the column.Example 21 eluted second.

Example 20. MS FAB-LSMIS) 462 [M+H]⁺

Example 21. Anal. Calculated for C₂₅H₂₀ClN₃O₄:C, 65.01; H, 4.36; N,9.10. Found C, 64.70; H, 4.06; N, 8.72.

EXAMPLE 22 Preparation of Compound XXII

Step 1

A solution of di-tert-butyl (2-hydroxyethyl)malonate (0.500 g, 1.92mmol), PPh₃ (0.555 g, 2.12 mmol) and CBr₄ (0.704 g, 2.12 mmol) in CH₂Cl₂(4.0 mL) was stirred at 0° C. for 5 min., then warmed to roomtemperature. After stirring for an additional 16 h, the reaction mixturewas concentrated in vacuo and purified via column chromatography (5-10%ethyl acetate-hexanes) to give 0.615 g (99%) of the desired product.TLC:R_(f)0.7 (silica, 10% EtOAc-hexanes).

Step 2

A flask containing 1,3-dihydro-5-methyl-2H-1,4-benzodiazepin-2-one(0.324 g, 1.03 mmol) and CS₂ CO₃ (0.900 g, 2.76 mmol) was dried undervacuum, flushed with Ar and charged with a solution of di-tert-butyl(2-bromoethyl) malonate (0.300 g, 0.929 mmol) in DMF (3.0 mL) at 0° C.The mixture was stirred at 0° C. for 15 min., room temperature for 15min., and 120° C. for 21 h. The reaction mixture was diluted with EtOAc(250 mL) and washed with water (2×50 mL). The organic layer wasseparated, dried over MgSO₄ and concentrated. Purification by columnchromatography (50-100% ethyl acetate-hexanes) afforded 0.017 g of thedesired product. TLC:R_(f)0.5 (silica, 100% EtOAc).

Step 3

A flask containing the mono alkylated malonate from step 2 (0.37 g) andsodium t-butoxide (0.009 g, 0.089 mmol) was vacuum dried, flushed withAr and diluted with THF (1.0 mL) at 0° C. After stirring at 0° C. for 30min., the reaction mixture was charged with4-bromoacetyl4′-chlorobiphenyl (0.027 g, 0.089 mmol) and subsequentlystirred at room temperature for an additional 5 h. The reaction mixturewas diluted with CH₂Cl₂ (75 mL) and washed with water (25 mL). Theorganic layer was separated, dried over MgSO₄ and concentrated. Crudepurification by column chromatography (50-100% ethyl acetate-hexanes)afforded the desired product (0.100 g, 0.154 mmol) which was useddirectly in step 4.

Step 4

A solution of the malonate from step 3 (0.100 g, 0.154 mmol) in formicacid (1.0 mL) was stirred at room temperature for 6 hrs. The resultingsolution was concentrated in vacuo and used directly in step 5.

Step 5

A solution of the product from step 4 in 1,4-dioxane (2.0 mL) was heatedto 100° C. for 16 h. After cooling to room temperate, the solvent wasremoved in vacuo. Purification by column chromatography (ethylacetate-hexanes-AcOH, 60:40:1) afforded 0.020 g of a mixture whichcontained the desired product. The mixture was purified via HPLC on aC18 column (acetonitrile-water) to furnish 2 mg of the target compound.HRMS 489.15720 (m+1), (calc. 488.15029).

EXAMPLE 23 Biological Assays of Invention Compounds

P218 Quenched Fluorescence Assay for MMP Inhibition

The P218 quenched fluorescence assay (Microfluorometric Profiling Assay)is a modification of that originally described by Knight, et al., FEBSLett. 296, 263 (1992) for a related substance and a variety of matrixmetalloproteinases (MMPs) in cuvettes. The assay was run with eachinvention compound and the three MMPs, MMP-3, MMP-9 and MMP-2, analyzedin parallel, adapted as follows for a 96-well microtiter plate and aHamilton AT® workstation.

P218 Fluorogenic Substrate

P218 is a synthetic substrate containing a 4-acetyl-7-methoxycoumarin(MCA) group in the N-terminal position and a 3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl (DPA) group internally. This isa modification of a peptide reported by Knight (1992) tat was used as asubstrate for matrix metalloproteinases. Once the P218 peptide iscleaved (putative clip site at the Ala-Leu bond), the fluorescence ofthe MCA group can be detected on a fluorometer with excitation at 328 nmand emission at 393 nm. P218 is currently being produced BACHEMexclusively for Bayer. P218 has the structure:

H-MCA-Pro-Lys-Pro-Leu-Ala-LeuDPA-Ala-Arg-NH2 (MW 1332.2)

Recombinant Human CHO Stromelysin (MMP-3)

Recombinant Human CHO Pro-MMP-3: Human CHO pro-stromelysin-257(pro-MMP-3) was expressed and purified as described by Housley, et al.,J. Biol. Chem. 2, 4481 (1993).

Activation of Pro-MMP-3: Pro MMP-3 at 1.72 μM (100 μg/mL) in 5 mM Trisat pH 7.5, 5 mM CaCl₂, 25 mM NaCl, and 0.005% Brij-35 (MMP-3 activationbuffer) was activated by incubation with TPCK (N-tosyl-(L)-phenylalaninechloromethyl ketone) trypsin (1:100 w/w to pro-MMP-3) at 25° C. for 30min. The reaction was stopped by addition of soybean trpsin inhibitor(SBTI;5:1 w/w to trypsin concentration). This activation protocolresults in the formation of 45 kDa active MMP-3, which still containsthe C-terminal portion of the enzyme.

Preparation of Human Recombinant Pro-Gelatinase A (MMP-2)

Recombinant Human Pro-MMP-2: Human pro-gelatinase A (proMMP-2) wasprepared using a vaccinia expression system according to the method ofFridman, et al., J. Biol. Chem. 267, 15398 (1992).

Activation of Pro-MMP-2: Pro-MMP-2 at 252 mg/mL was diluted 1:5 to afinal concentration of 50 μg/mL solution in 25 mM Tris at pH 7.5, 5 mMCaCl₂, 150 mM NaCl, and 0.005% Brij-35 (MMP-2 activation buffer).p-Aminophenylmercuric acetate (APMA) was prepared in 10 mM (3.5 mg/mL)in 0.05 NaOH. The APMA solution was added at {fraction (1/20)} thereaction volume for a final AMPA concentration of 0.5 mM, and the enzymewas incubated at 37° C. for 30 min. Activated MMP-2 (15 mL) was dialyzedtwice vs. 2 L of MMP-2 activation buffer (dialysis membranes werepre-treated with a solution consisting of 0.1% BSA in MMP-2 activationbuffer for 1 min. followed by extensive H₂O washing). The enzyme wasconcentrated on Centricon concentrators (concentrators were alsopre-treated a solution consisting of 0.1% BSA in MMP-2 activation bufferfor 1 min.. followed by washing with H₂O, then MMP-2 activation buffer)with re-dilution followed by re-concentration repeated twice. The enzymewas diluted to 7.5 mL (0.5 times the original volume) with MMP-2activation buffer.

Preparation of Human Recombinant Pro-Gelatinase B (MMP-9)

Recombinant Human Pro-MMP-9: Human pro-gelatinase B (pro-MMP-9) derivedfrom U937 cDNA as described by Wilhelm, et al. J. Biol. Chem. 264, 17213(1989) was expressed as the fill-length form using a baculovirus proteinexpression system. The pro-enzyme was purified using methods previouslydescribed by Hibbs, et al. J. Biol. Chem.260, 2493 (1984).

Activation of Pro-MMP-9: Pro-MMP-2 20 μg/mL in 50 mM Tris at pH 7.4, 10mM CaCl₂, 150 mM NaCl, and 0.005% Brij-35 (MMP-9 activation buffer) wasactivated by incubation with 0.5 mM p-aminophenylmercuric acetate (APMA)for 3.5 h at 37° C. The enzyme was dialyzed against the same buffer torevmove the APMA.

Instrumentation

Hamilton Microlab AT Plus: The MMP-Profiling Assay is performedrobotically on a Hamilton MicroLab AT Plus®. The Hamilton is programmedto: (1) serially dilute up to 11 potential inhibitors automatically froma 2.5 mM stock in 100% DMSO; (2) distribute substrate followed byinhibitor into a 96 well Cytofluor plate; and (3) add a single enyme tothe plate with mixing to start the Bron. Subsequent plats for eachadditional enzyme are prepared automatically by beginning the program atthe substrate addition point, remixing the diluted inhibitors andbeginning the reaction by addition of enzyme. In this way, all MMPassays were done using the same inhibitor dilutions.

Millipore Cytofluor II. Following incubation, the plate was read on aCytofluor II fluorometric plate reader with excitation at 340 nM andemission at 395 nM with the gain set at 80.

Buffers

Microfluorometric Reaction Buffer (MRB): Dilution of test compounds,enzymes, and P218 substrate for the microfluorometric assay were made inmicrofluorometric reaction buffer consisting of 50 mM2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.5 with 10 mM CaCl₂,150 mM NaCl, 0.005% Brij-35 and 1% DMSO.

Methods

MMP Microfluorometric Profiling Assay. The assay is done with a finalsubstrate concentration of 6 μM P218 and approximately 0.5 to 0.8 nM MMPwith variable drug concentrations. The Hamilton is programmed toserially dilute up to 11 compounds from a 2.5 mM stock (100% DMSO) to10x the final compounds concentrations in the assay. Initially, theinstrument delivers various amounts of microfluoromentric reactionbuffer (MRB) to a 96 tube rack of 1 ml Marsh dilution tubes. Theinstrument then picks up 20 μl of inhibitor (2.5 mM) from the samplerack and mixes it with a buffer in row A of the Marsh rack, resulting ina 50 μM drug concentration. The inhibitors are then serially diluted to10, 5, 1, 0.2, 0.05 and 0.01 μM. Position 1 on the sample rack containsonly DMSO for the “enzyme-only” wells in the assay, which results in noinhibitor in column 1, rows A through H. The instrument then distributes107 μl of P218 substrate (8.2 μM in MRB) to a single 96 well cytofluormicrotiter plate. The instrument re-mixes and loads 14.5 μl of dilutedcompound from rows A to G in the Marsh rack to corresponding rows in themicrotiter plate. (Row H represents the “background” row and 39.5 μl ofMRB is delivered in placed of drug or enzyme). The reaction is startedby adding 25 μl of the appropriate enzyme (at 5.86 times the finalenzyme concentration) from a BSA treated reagent reservoir to each well,excluding Row H, the “background” row. (The enzyme reservoir ispretreated with 1% BSA in 50 mM Tris,. pH 7.5 containing 150 mM NaCl for1 hour at room temp., followed by extensive H₂O washing and drying atroom temp.).

After addition and mixing of the enzyme, the plate is covered andincubated for 25 min. at 37° C. Additional enzymes are tested in thesame manner by beginning the Hamilton program with the distribution ofP218 substrate to the microtiter plate, followed by re-mixing anddistribution of the drug from the same Marsh rack to the microtiterplate. The second (or third, etc.) MMP to be tested is then distributedfrom a reagent rack to the microtiter plate with mixing, prior tocovering and incubation. This is repeated for all additional MMP's to betested.

IC50 Determination in Microfluorometric Assay: Data generated on theCytofluor II is copied from an exported “.CSV” file to a master Excelspreadsheet Data from several different MMPs (one 96 well plate per MMP)were calculated simultaneously. The percent inhibition is determinationfor each drug concentration by comparing the amount of hydrolysis(fluorescence units generated over 25 minutes of hydrolysis) of wellscontaining compound with the “enzyme only” wells in column 1. Followingsubtraction of the background the percent inhibition was calculated as:

((Control values−Treated values)/Control values)×100

Percent inhibitions were determined for inhibitor concentrations of 5,1, 0.5, 0.1, 0.02, 0.005 and, 0.001 μM of drug. Linear regressionanalysis of percent inhibition versus log inhibitor concentration wasused to obtain IC₅₀ values.

TABLE 2 Exam- MMP-3 Fluorogenic MMP-9 Fluorogenic MMP-2 Fluorogenic pleIC₅₀ nm IC₅₀ nm IC₅₀ nm 1 1.7 0.34 0.39 2 17 24 9.5 3 31 67 21 4 9.2 2.14.2 5 4.2 2.3 1.4 6 4.1 4.3 0.5 7 14 110 10 8 2.0 6.2 1.0 18 59 32 13 1947 4.7 2.4 20 320 84 57 21 6.5 2.1 1.5 22 140 120 24

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

We claim:
 1. A matrix metalloprotease inhibitor having the formula:

wherein r is 0-2, T is selected from the group consisting of:

and R⁴⁰ represents


2. A composition having matrix metalloprotease inhibitory activity,comprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 3. A method of inhibiting matrix metalloprotease activity in amammal comprising administration of an effective amount of matrixmetalloprotease inhibitor compound of claim 1 to said mammal.
 4. Themethod of claim 3 wherein said mammal is a human.
 5. A method oftreating a mammal comprising administering to the mammal a matrixmetalloprotease inhibiting amount of a compound according to claim 1sufficient to: (a) alleviate the effects of osteoarthritis, rheumatoidarthritis, septic arthritis, periodontal disease, corneal ulceration,proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis,bullosa, conditions leading to inflammatory responses, osteopeniasmediated by MMP activity, tempero mandibular joint disease, demyelatingdiseases of the nervous system; (b) retard tumor metastasis ordegenerative cartilage loss following traumatic joint injury; (c) reducecoronary thrombosis from athrosclerotic plaque rupture; or (d) effectbirth control.
 6. The method of claim 5 wherein the effect isalleviation of osteoarthritis.
 7. The method of claim 5 wherein theeffect is retardation of tumor metastasis.